The air pumps make a loud noise and lithium ion batteries require care when handling. Roses have sharp prickles, wear gloves to protect from scratches. Hearing protection is recommended. There may be a risk of electric shock in rainy conditions. Wear appropriate protection from the sun: sunscreen and water are recommended.
For this experiment, we will be collecting headspace volatiles with Solid Phase Microextraction (SPME), a method which adsorbs the volatile organic compounds (VOCs) surrounding part of a plant to the SPME fiber. The adsorbed chemicals can then be desorbed with paired Gas Chromatography - Mass Spectrometery (GC-MS) equipment to identify the collected chemistry. SPME extraction is a flexible method which can collect the VOCs from just about anything you can get an container around. For our experiments, we rely on heat-treated oven bags, which are relatively chemically inert after cooking them in an oven at 50°C for a day or so.
The purpose of these experiments is to collect the volatiles emanating from roses under various conditions, including:
By sampling rose VOCs with SPME under these different conditions, we hope to find quantitative and/or qualitative differences in VOC compounds between rose treatments.
Our hypothesis formed from preliminary research where we observed that A. swirskii were preferentially attracted the RRD-infected roses. We think this might be due to differences in the VOCs released from uninfected vs. infected roses. Collecting and comparing chemistry between different roses should allow us to see any qualitative and/or quantitative differences in VOCs released between these groups. This data will give us insight into some of the metabolic changes which may have occurred between different rose groups. Furthermore, our data should indicate which chemicals are worth testing for attractiveness to A. swirskii in olfactometer trials, which can give us insight into which specific VOCs A. swirskii may have been responding to from the RRD-infected roses. There is also a secondary assumption involved: it is assumed that A. swirskii’ may be associating these changes in VOCs with their prey–herbivorous mites–which is why they are attracted to RRD-infested roses. Infected roses have populations of Phyllocoptes fructiphilus, the vector of RRD. Although these mites generally are inaccessible to A. swirkii (due to their cryptic habits of living among the dense trichomes present on the adaxial surfaces of rose sepals), both mite and virus likely reduce/change plant defenses. Reducing plant defenses in many cases is advantageous for other herbivores feeding on the infected plants and increase their populations. It might be that predatory mites are more successful at finding prey due to larger herbivore populations present on infected roses. This is where the Acibenzolar-S-Methyl (ASM, Actigard) treatments come in: ASM is an analogue of Methyl Salicylate (MeSA), a chemical which activates Systemic Acquired Resistance (SAR), part of a plant’s immune response to pathogen attack. The SAR pathway releases a number of chemicals which are involved with a plant’s ability to combat pathogens and fungi, as well as effects on predatory mites and herbivorous mite biology and behavior. Specifically, MeSA has been reported to be attractive to some predatory mites, and we would expect to see changes in the levels of MeSA released by RRD-infected plants as well as plants which have been induced with ASM.
Herbivorous mites tend to be found on the tender plant tissues found on actively growing parts of the rose, as well as flowers. With this in mind, we want to select rose canes with flowers, where the prey of A. swirskii are likely to be found. Selecting a flowering can also collects a greater diversity of VOCs than we would otherwise if we only sampled leaf or cane tissues. Although the actual VOC extraction takes only around 20 minutes, the entire process requires at least an hour of wait time to allow VOCs to concentrate in our oven bags before we can begin SPME. Our lab has two SPME holders available, so two extractions can be done at once. Temperature has a large effect on VOC collection, so it is in our best interest to do as many as many SPME extractions as possible during the same field visit to minimize environmental variables. Also, it is important to record the date of extractions so we can collect and report weather information for that day. The SPME fibers are fragile, and can break if the needle becomes excessively bent: take care to guide the septum piercing needle as you perforate the bag for VOC extraction. Be diligent in remembering to uncock the plunger when you are done with extraction/injection; removing the needle with the fiber exposed can break it. Lastly, take care to ensure that the fiber does not come into contact with the rose during extraction.